Kim Geon-Woo, Lee Seung-Hoon, Cho Hee, Kim Minwoo, Shin Eui-Cheol, Oh Jong-Won
Department of Biotechnology, Yonsei University, Seoul, Korea.
Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea.
PLoS Pathog. 2016 Jul 1;12(7):e1005714. doi: 10.1371/journal.ppat.1005714. eCollection 2016 Jul.
The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3' end by 3'-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3'-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3' end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation.
肝脏特异性微小RNA miR-122在肝脏发育和代谢中起重要作用，是丙型肝炎病毒（HCV）的关键前病毒因子。尽管其在肝脏和HCV生命周期中起关键作用，但关于HCV感染对miR-122表达调控的分子机制知之甚少。在此，我们表明HCV核心蛋白通过抑制GLD-2（一种非经典细胞质聚腺苷酸聚合酶）促进miR-122的去稳定化，从而下调miR-122的丰度。miR-122表达的降低导致miR-122已知功能的失调，包括其对HCV的前病毒活性。通过对人类肝脏活检组织中的小RNA进行高通量测序，我们发现22个核苷酸（nt）的原型miR-122在其3'端通过3'端非模板化和模板化核苷酸添加进行修饰。值得注意的是，在HCV患者的肝脏标本中，带有任何核糖核苷酸单核苷酸尾巴的miR-122异构体比例降低。我们发现这些单核苷酸尾巴的miR-122异构体比22-nt原型miR-122表现出更高的miRNA活性和稳定性，并且3'端延伸由GLD-2独特的末端核苷酸转移酶活性催化，该酶能够在不偏好腺苷酸的情况下将任何单个核糖核苷酸添加到miR-122的3'端。HCV核心蛋白特异性抑制GLD-2，并且发现其在细胞质中与GLD-2的相互作用是miR-122下调的原因。总的来说，我们的结果为HCV核心蛋白通过调节miR-122稳定性来控制病毒RNA丰度和miR-122功能的调控作用提供了新的见解。
